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International audience; A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length poly-morphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping. The intestinal protozoan Giardia lamblia (synonyms, G. in-testinalis and G. duodenalis [1]) is a cosmopolitan parasite frequently involved in human parasitic gastroenteritis throughout the world. Transmission of the G. lamblia cyst to humans occurs mainly following ingestion of contaminated water. Clinical manifestations of symptomatic giardiasis include greasy stools, flatulence, diarrhea, and abdominal cramps (9). However , the majority of cases are asymptomatic or minimally symptomatic in immunocompetent individuals. Among the six species identified in the Giardia genus, only G. lamblia infects humans and numerous other mammals as well (1, 25). Moreover, isolates of G. lamblia are classified into seven assemblages, based on the characterization of the gluta-mate dehydrogenase (gdh), small-subunit (SSU) rRNA, and triosephosphate isomerase (tpi) genes (12, 18, 20, 21). Assemblages A and B infect humans and a broad range of other hosts, including livestock, cats, dogs, and wild mammals. The assemblage A isolates have been further grouped into subgroups I and II. The assemblage B isolates have been separated into subgroups III and IV (17, 24). Genetic assemblages C, D, E, F, and G appear to be host restricted to domestic animals, livestock , and wild animals (19, 21). At present, antigen detection immunoassays for Giardia are used as the routine diagnostic procedure of choice in many hospitals and public health laboratories (8, 13, 27). However, these methods are unable to differentiate between the genetic asemblages of Giardia lamblia. Molecular detection methods based on PCR have been developed to detect G. lamblia cysts in feces. These techniques have numerous advantages in terms of sensitivity, speed, and specificity in comparison to conventional methodologies (3, 16). Moreover, these molecular techniques may allow the genotyping of Giardia lamblia cysts (3, 4, 20). We previously described a method for the successful extraction and detection of giardial DNA from naturally contaminated wastewater (5). In the present study, we evaluated primers for rapid and sensitive classification of G. lamblia cysts from human feces into assemblages A and B. These primers were previously designed for the detection and quantification of G. lamblia assemblages A and B in environmental samples by real-time PCR (data not shown). In this study, we evaluated the distribution of these major assemblages in sporadic human giardiasis in France and also in samples from 15 French soldiers coming back from the Ivory Coast. Moreover, the simultaneous use of our assay and two previously described PCR-restriction fragment length polymorphism (RFLP) assays allowed comparison of the tpi and gdh genes for detection and genotyping of G. lamblia. MATERIALS AND METHODS Purified suspensions. Giardia lamblia assemblage B cysts, produced by passage of human strain H3 (10) of G. lamblia through Mongolian gerbils, were obtained from Waterborne Inc. (New Orleans, La.). Prior to delivery, cysts were 95 to 99% purified by sucrose and Percoll density gradient centrifugation and washing with water, and then stored in phosphate-buffered saline (PBS, pH 7.4) with antibiotics. Giardia lamblia (assemblage A) cysts were purified from one human feces sample by the ethyl acetate procedure (see below) followed by * Corresponding author. Mailing address: LCPME-UMR 7564 CNRS-UHP, Equipe Microbiologie et Physique, Faculté de Pharmacie

Dans la région de Marrakech, les eaux usées sont utilisées pour l'irrigation sans aucun traitement. Par ailleurs ces eaux usées ruissellent dans des canaux à ciel ouvert sur des distances supérieures à 2 km. Nous avons étudié le devenir des kystes de protozoaires et des oeufs d'helminthes sur le parcours d'un canal alimenté par un émissaire d'eau usée (E2). L'analyse parasitologique des eaux usées a montré que ces eaux contiennent plusieurs types de parasites. En effet, nous avons observé la présence des kystes de protozoaires (Entamoeba histolytica, Entamoebacoli, Giardiasp.) et des oeufs d'helminthes pathogènes (Ascaris,Trichuris, Enterobius, Hymenolepis...). L'étude de la charge parasitaire le long du cours d'eau usée montre une diminution du nombre de parasites dans l'eau usée en allant de l'embouchure S0 vers l'aval de l'émissaire E2. Avec des concentrations moyennes de 1,6.105 kystes/L et 145 oeufs/L à S0, alors qu'à 2 km de S0 (SV) il n'est plus retrouvé que 2,7.104 kystes/L et 33,4 oeufs/L. La charge parasitaire dans le sédiment du cours d'eau, contrairement à l'eau, augmente en s'éloignant de l'embouchure S0. Ainsi, les concentrations moyennes de kystes de protozoaires et les oeufs d'helminthes sont respectivement de 1,7.104 kystes/g et de 9,6 oeufs/g à 80 m de S0 et atteignent, 1,4.105 kystes/g et 78,1 oeufs/g à 2 km de S0. Le ruissellement des eaux usées sur un parcours de 2 Km, à ciel ouvert, montre des taux d'abattement importants des parasites. En effet, plus de 83 % des kystes de protozoaires et plus de 77 % des oeufs d'helminthes sont éliminés de l'effluent. Mais ces abattements restent insuffisants pour satisfaire les directives de l'OMS (1989) en matière de réutilisation des eaux usées en agriculture., The use of wastewater in agriculture in what is called "sewage farming" is becoming more widespread, particularly in countries with a severe shortage of water resources. Wastewater from the city of Marrakech is used for irrigation without any treatment. Nevertheless, the wastewater runs into an open channel of 2 km before being used. Thus, the fate of helminth eggs and protozoan cysts in this channel is of great importance for public health. The effluent studied carries wastewater from highly populated residential areas (Sidi Youssef Ben Ali, la Médina...). The primary channel originating from this effluent runs for 2 km with a variable width of 0.7 to 5.5 m in a clay soil. Secondary ramifications on the channel are used for irrigation. Water and sediment samples were collected at different locations on the channel twice monthly for five months (April-August). One litre wastewater samples and 10 g sediment samples were collected, at all stations on the channel. The samples were analysed using the concentration method of Teleman-Rivas as modified by Bailenger (1962) because of its reliability and low cost. Helminth eggs were then counted on MacMaster cells after addition of saturated saccharose solution to the samples. Protozoan cysts were quantified using a Thoma cell after addition of Lugol solution to the samples.Results of water analyses are expressed as the number of cysts or eggs per liter of wastewater and results for sediments are expressed as the number of cysts or eggs per gram of dry matter.- Wastewater samples from the effluent contained helminths as well as protozoa. All wastewater samples contained protozoan cysts. Among three species of protozoa identified, two were pathogenic: Entamoebahistolytica, Giardiasp. and one saprophytic Entamoebacoli. All water samples contained helminth eggs. Analyses for helminth egg revealed the presence of Nematodes (Ascaris, Trichuris, Enterobius) and Cestodes (Hymenolepis, Moniezia). This results mainly from Ascaris eggs that were encountered in all water samples. The concentration of helminth eggs in the sewage was much less than the protozoan cysts. In terms of densities, protozoan cysts were in the range 6.9 x 10[exp]4 to 2.0 x 10[exp]5 cysts /L with an average of 1.6 x 10[exp]5 cysts/L. Pathogenic protozoa represent 49 % of total cysts. The wastewater at the beginning of the channel contained from 102 to 238 helminth eggs/L with an average of 145 helminth eggs/L. Nematode eggs represented 72 % of total helminth eggs. Ascaris eggs dominated in the water samples with 75.6 eggs/L representing 52 % of total helminth eggs. The densities of protozoan cysts as well as helminth eggs in wastewater samples decreased dramatically along the open channel. Protozoan cysts dropped from 1.6 x 10[exp]5 cysts/L at the beginning of the channel to 2.7 x 10[exp]4 cysts/L at a distance 2 km in the open channel. Pathogenic protozoa decreased from 8.1 x 10[exp]4 cysts/L to 1.0 x 10[exp]4 cysts/L for the same distance. The average of helminth eggs also dropped from 145 eggs/L to 33 eggs/L. Ascaris eggs showed the highest decrease from 76 eggs/L to 12 eggs/L. The genus Trichuris showed the lowest decrease from 11 eggs/L to 6.8 eggs/L. - Analyses of sediment samples revealed the presence of all protozoan cysts and helminth eggs. Contrary to water samples, sediments samples showed an increase of all egg and cyst concentrations along the open channel. Protozoan cysts progressively increased from 1.7 x 10[exp]4 cysts/g at 80 m in the open channel to 1.4 x 10[exp]5 cysts/g at 2 km distance in the channel. Helminth parasites also increased from 9.6 eggs/g to 78 eggs/g for the same distance. This increase of helminth eggs and protozoan cysts resulted from the decrease of water flow velocity which caused their sedimentation. Parasites settle out of the water column because of their own weight and because they adsorb to particles accelerating their settling. Among all parasites Ascaris eggs settle out first before all other nematode eggs. Ascaris eggs were detected at 80 m in the open channel while Enterobius eggs were not observed until 560 m in the channel. Trichuris eggs were not observed in any sediment sample. Trichuris eggs are more easily carried by the water flow because of their hydrodynamic fusiform shape which may explain their absence in sediment samples. In conclusion, the parasitic quality of a domestic wastewater in Marrakech showed significant improvement after 2 km in an open channel. Both helminth eggs and protozoan cysts showed significant removal percentage of 77 % and 83 % after 2 km running in the open channel. The best percent removal of protozoan cysts was observed for pathogenic protozoa with 88 % for Entamoeba histolytica and 87 % for Giardia sp. For helminth eggs, Ascaris eggs showed the highest removal percentage with 85 % while Trichuris showed the lowest with 37 %. However, the concentration of helminth eggs and protozoan cysts, after the sewage flows over a distance of 2 km in the open channel, are still higher than the guidelines of the W.H.O. (1989) for the use of wastewater in agriculture (less than one nematode egg per litre). These wastewaters require further treatment before they can be used for irrigation.

The presence of helminth eggs (Ascaris eggs) in sewage sludge may constitute a sanitary risk when used as agricultural fertiliser. Sanitisation of sewage sludge can be achieved by treatment with quick lime, a process that destroys sludge pathogens in two ways: pH increase and temperature rise. Among the pathogens of epidemiological relevance, Ascaris eggs are the most resistant to liming, and, hence, may serve as indicators of hygienic quality of biosolids. This research aims at defining, between 50 degrees C and 60 degrees C, the time required in the case of limed sludge to obtain a product with a negligible level of viable Ascaris eggs. To achieve this objective, investigations on inactivation kinetics of Ascaris eggs were conducted in the following products: contaminated milk of lime; naturally contaminated sludge treated with slaked lime and heat; naturally contaminated sludge treated with quick lime; and sludge treated at full scale with quick lime. For the inactivation kinetics where a negligible level of Ascaris eggs was reached, the inactivation threshold was determined. Depending on the experimental situation, the inactivation threshold period was found to fluctuate between 5 and 75 min at 55 degrees C and between 1 and 8 min at 60 degrees C.

This study was performed on sludge samples from 20 wastewater treatment plants located in the north west of France with capacities of 1,000-20,000 inhabitant equivalents. The types of treatment studied were activated sludge low charge with and without denitrification. Respectively, 110 samples of fresh sludge and 84 samples of discharged sludge for spreading were analysed. Globally 78.6% of samples contained helminth eggs belonging to the cestodes (6.1%) and nematodes (93.9%). Most of the nematode eggs detected were viable with 135 positive samples. The distribution, according to genera, indicated a high prevalence of Toxocara eggs (77.4%) followed by Capillaria (13.2%), Trichuris (8.1%) and Ascaris (1.3%). For viable nematode eggs, the concentrations detected ranged from

The purpose of this study was to improve understanding of the potential for transfer of the protozoan pathogen Cryptosporidium parvum through aquifers to drinking water wells. Therefore, the factors characterising this transport were experimentally determined. We have developed a continuously recirculating column assay. The latter allows small amounts of C. parvum oocysts to be manipulated providing as much protection as possible from the risks of contamination. As the analysis of oocyst samples is time consuming, a numerical model, simulating the transport phenomena of oocysts under the experimental conditions of assays, was developed to establish the whole experimental curve of results using a small number of experimental points. The comparisons drawn between analytic solutions, experimental results with tracer (NaCl solution) and numerical simulation were in good agreement. A continuously recirculating column assay was performed using oocysts in suspension (flow rate = 1.43 mL/min). Treated sand was used as previous experiments had shown that no adsorption occurs. We observed almost total filtration (99.85%). To check this result, an assay with an open column was carried out under the same conditions. We observed a filtration value of 97%. Consequently, we may say that the continuously recirculating column assay provides satisfactory results.

International audience; We investigated the efficiency of radiation treatment to destroy Ascaris ova viability and found that no ova were viable after exposure to 0.75 kGy (D90 at 0.39 kGy). Although the outer coat of the Ascaris ovum had a protective effect at low doses (0.20 kGy) no difference in ova viability was observed at 0.30 kGy. As the doses commonly used for sanitary treatment of wastewater are much higher, Ascaris ova should be effectively eliminated from sludge by the 10 kGy dose required by EPA regulations (U.S. EPA, 1993 Federal Register 58, 9248–9415).

The goal of this work was to quantify the effectiveness of wastewater treatment systems in removing Giardia cysts. For this, the quantity and the viability of the cysts recovered from primary and digested sludge were studied. The digestion treatment has no effect on the number of cysts recovered, however it affects their viability.

The present study consists of assessing the efficiency of a macrophyte (Phragmites australis) system in wastewater purification in an arid climate. The experimental system is made up of three beds differing in length (30, 40 and 50 m) and implanted with Phragmites australis. Wastewater inflow through these beds is horizontal with a flow of 10 l.s−1. Retention time varies between 1 and 4 h. The frequency of irrigation is weekly. The studied parameters are: the organic load (COD and TSS), nutrients (total Kjeldhal nitrogen: TKN and total phosphorus: TP) and the parasitical load (helminth eggs). Water, soil and plants are the three components of the experimental system we analyzed. Results reveal that in the output, the decrease in organic load, nutrients and parasitical load is very sharp in the large bed (COD: 62%, TKN: 43%, TP: 14%, helminth eggs: 93%). However, in the small bed (30 m), the decrease is slight (COD: 48%, TKN: 23%, TP: 5%, helminth eggs: 88%). Concerning parasitical load, the study shows that the three beds are effective in eliminating helminth eggs, even with a very short retention time (1–4 h). The quality of the effluent of the reed bed system should be classified within category B according to WHO guidelines (1989) for crops irrigation. Soil sample analysis from entrance to exit shows a decreasing gradient of nitrogen (N), phosphorus (P), organic carbon (Org.C), organic matter (OM) and parasitical helmith eggs. The gradient becomes more significant in the largest bed with decreasing rate, reaching respectively 49%, 44%, 37%, 40% and 100% for TKN, TP, Org.C, OM and helminth eggs. As for the plant material, the composition of major elements (nitrogen and phosphorus) of Phragmites aerial parts shows the same evolution. While the decreasing rate of nitrogen in the leaves reaches 50% in the largest bed, it does not exceed 8% in the smallest bed. Mean aerial productivity of the studied reed beds was estimated at 134 tons dry weight.ha−1. The aerial biomass exported contains only 5–6% of nitrogen and 10–12% of phosphorus with regard to their load at the influent.

Microbial adhesion to hydrocarbons and microelectrophoresis were investigated in order to characterize the surface properties of Cryptosporidium parvum. Oocysts exhibited low removal rates by octane (only 20% on average), suggesting that the Cryptosporidium sp. does not demonstrate marked hydrophobic properties. A zeta potential close to -25 mV at pH 6 to 6.5 in deionized water was observed for the parasite. Measurements of hydrophobicity and zeta potential were performed as a function of pH and ionic strength or conductivity. Hydrophobicity maxima were observed at extreme pH values, with 40% of adhesion of oocysts to octane. It also appeared that ionic strength (estimated by conductivity) could influence the hydrophobic properties of oocysts. Cryptosporidium oocysts showed a pH-dependent surface charge, with zeta potentials becoming less negative as pH was reduced, starting at -35 mV for alkaline pH and reaching 0 at isoelectric points for pH 2.5. On the other hand, variation of surface charge with respect to conductivity of the suspension tested in this work was quite small. The knowledge of hydrophobic properties and surface charge of the parasite provides information useful in, for example, the choice of various flocculation treatments, membrane filters, and cleaning agents in connection with oocyst recovery.

This paper presents results of two years of investigations on three types of wastewater stabilization pond treatment systems, purifying a raw urban wastewater, in the arid climate of Marrakesh. The systems tested were: two lined water hyacinth ponds, two lined facultative ponds and one anaerobic pond. During the course of the experiment, organic load, nutrients and parasitical load were studied. Results show that the macrophytic ponds were more efficient to reduce organic load (90% of TSS and 78% of COD) that the microphtic ponds; these ones were more efficient to eliminate nutrients (NTK 71%, NH4 60%, Ptotal 80% and PO4 62%). The anaerobic pond presents a lower efficiency, less than 40% for organic and less than 20% for nutrients. About sanitary concerns, all of the year, the macrophytic and microphytic pond effluents correspond to B category (WHO, 1989). The anaerobic pond yields B category effluent except in autumn when it corresponds to C category.

The development of an extraction-concentration method for the nematode eggs existing in urban treatment-plant sludge made it possible to study the viable nature of these eggs by cultivating them in deionized water at 30°C for 30 days. The criterion selected to show viability was the development of eggs at the larval stage. Sludge samples from various origins (physical, biological and chemical treatments) were analyzed: 93% of the eggs proved to be viable in primary sedimentation sludge. Eggs that can grow have been detected in samples from biological treatments, with, however, a lower frequency for systems calling for lagooning and compositing with, respectively, 26 and 25% of viable eggs. Anaerobic digestion gave intermediate results with 66%, whereas a prolonged aeration led to a limited effect, 93% of eggs being considered as viable. A 15 day chemical treatment with lime did not produce complete elimination of viable eggs: the analysis showed 66% of eggs developing at the larval stage. This study thus confirmed that nematode eggs are strongly resistant to most of the classical waste treatments.

Waste-water from Ben Sergao (Morocco) is treated by a process using infiltration on sand which includes an anaerobic lagoon (1500 m3) in which waste-water has a retention time of 1.4 days followed by 5 infiltration basins (1500 m2 each). Each basin comprises by a 2 meter thick sand dune layer. The basins are drained at their base through a network of gravel-embedded drains. Three 350 m3 batches are performed daily. These are received in 3 separate basins. Each basin receives one batch a day during 3 days and is then left to rest for 2 days before carrying out a new cycle. It has been estimated that the treatment induced a decrease of 99.93 % for the fecal coliforms and 99.98 % for the fecal streptococci. Concerning the parasitology, neither nematode eggs nor cestode eggs were found in treated sewage even though their number in raw waste-water could be estimated respectively to be 139 and 75 per liter. As for physicochemical parameters, no significant difference appears between raw and treated sewage for K, P and conductivity, but there is a very significant increase of parameters NO2 and NO3 and a very important decrease of COD and total suspended matter after treatment. This treatment process seems to be very well adapted for the treatment of sewage and the reuse of treated water in rural areas.

In countries with limited water resources, the agricultural use of treated wastewater represents an interesting alternative. The recent World Health Organisation guidelines (1989) for the microbiological quality of treated wastewaters used for crop irrigation require an arithmetic mean of ≤ 1 intestinal nematode egg per litre. In this study diverse methods have been tried for recovering parasitic nematode eggs from vegetables artificially contaminated by Ascaris eggs. The vegetables examined were lettuce and tomatoes. Vegetables were seeded with Ascaris eggs at different levels from 3 to 900 eggs. Eggs elution was realized with chemical solution (distilled water, aceto-acetic buffer pH 4.5, formalin 20%, anionic detergent DDN50®, cationic detergent hyamine®) and/or mechanical treatment (brush - scrub or brush - scrub coupled with glass powder treatment). Concentration was performed by flotation technique with a 55% zinc sulphate solution (d=1.3). Recovery was low for all samples after chemical elution. When we tested the association of chemical and mechanical treatments the percent recovery were ranging from 72 to 100% for vegetables contaminated with 10 to 400 eggs. The method proposed includes an elution with a glass powder suspension in a cationic detergent (50/50), a concentration with ZnSO4 55% solution (d=1.3) and a quantification with a counting cell. The sensitivity threshold of the proposed method lies at about 10 eggs per samples.

Various Campylobacter isolation methodologies from wastewater samples have been tested. Considering the results obtained, an experimental method is suggested combining a filtration on a 0.45 µm membrane with an enrichment in Preston's broth with 6 h at 42°C of incubation followed by an isolation on Preston's agar after a 48 hours incubation at 42° C in a microaerophilic atmosphere. This technique has been applied to 116 samples of raw wastewater taken from the Nancy treatment plant. Campylobacter have been isolated in 43.9 % of the samples. The species distribution was 94.1 % C. jejuni and 5.9 % C. coli.

Cryptosporidium a ete retrouve dans differents types d'eaux (usees, surface, traitees) a des concentrations et des taux d'isolement consequents.De plus, il affiche une resistance tres importante vis-a-vis des differents systemes utilises dans les stations de traitement et les differents modes de desinfection appliques dans les reseaux.Le controle de Cryptosporidium dans les eaux est donc ressenti aujourd'hui comme un imperatif notoire.Une revue de la litterature a ete realisee a propos des techniques appropriees aux differents types d'eaux et aux mises en evidence de ce protozoaire dans l'eau potable.Une application a ete developpee dans le cadre de la recherche de Cryptosporidium a partir d'eaux usees par filtration sur cartouche de polypropylene.

Giardia intestinalis is a protozoon which has generated numerous waterborne outbreaks of giardiasis in the United States. In this study Giardia cysts were searched in raw wastewater after concentration by Bailenger method and quantification with Thoma cell counting. Different parameters were studied : sampling time, sampling day, sampling month. The study of hourly variations shows concentration of Giardia cysts ranged from 9.5 × 103 to 1.4 × 104/l with a significant difference between the samples taken at 10 a.m. and those taken in the afternoon. No significant daily variation has been shown in the mean value of the 6 samples taken every day. The results of monthly variations indicate that it is in February (5.9 × 103/l) and mainly in March (1.2 × 104/l) that the concentrations of the Giardia cysts are the highest. The lowest values are noticed in November (1.3 × 103/l).

In countries with limited water resources, the agricultural use of treated wastewater represents an interesting alternative. A recently published international report (Engelberg report 1985) shows the importance of strict parasitological criteria for such reuse. In this study we have tested the survival of Ascaris eggs on different edible crops (lettuce, radishes, chives) and in different soils (clay, sand, silt) irrigated with water artificially contaminated. Leaves and roots from vegetables, surface and deep samples from soil were analyzed. In the soil the survival of Ascaris eggs is dependent on 2 factors : exposure to sunlight and soil type. Up to 20 days the Ascaris egg survival is significant,after which time it decreases quickly. In vegetable samples 10 days after the contamination by spraying no eggs are found on leaves of any crops. In roots samples after 10 days the Ascaris recovery is dependent on type of vegetables. These differences could be explained by different radicular system. After 45 days, the decrease is similar for all the crops. These results underline the importance of parasitological criteria for the agricultural wastewater reuse.

La contamination parasitaire des boues residuaires a ete evaluee sur differents types de prelevements.. L'analyse de 116 echantillons de boues primaires digerees et semi deshydratees a montre que tous les echantillons renfermaient des oeufs d'helminthes a des concentrations variant de 113 a 135/100g de boues.. Au niveau des boues mises en decharge le nombre d'oeufs retrouve varie considerablement en fonction du temps de sejour. Si aucun oeuf n'a pu etre mis en evidence dans les boues placees en decharge depuis 4 ans, en revanche dans les boues ayant sejourne de 1 a 3 ans, des concentrations moyennes de 12 oeufs/100g sont observees.. Pour les boues rejetees en mer, l'etude des sediments marins dans la zone de rejet montre l'importance des oeufs d'helminthes dans la contamination microbiologique des sediments.. Enfin l'etude de 60 echantillons de compost constitues de boues residuaires et d'ordures menageres montre que 60% des prelevements renfermaient des oeufs d'helminthes.

International audience; Among the seven assemblages identified in Giardia duodenalis species, only assemblages A and B infect humans and numerous other mammals as well. On the other hand, assemblage E is considered to be host restricted to livestock. The aim of the present study was to compare the presence of G. duodenalis assemblages A, B and E in wastewater samples from two municipal treatment plants (n=24) and one slaughterhouse (n=12). Thus, PCR assays targeting the tpi gene were developed to detect specifically these three G. duodenalis assemblages. Assemblages A and B were detected in urban wastewater with a predominance of assemblage A, especially for one treatment plant. Concerning slaughterhouse wastewater, assemblage A was found in 58% of the samples, whereas assemblage B was not detected. Assemblage E was not detected in urban wastewater, but was found in 92% of the samples from slaughterhouse. Thus, combination of assemblages A and B seemed to indicate a human contamination origin, while combination of assemblages A and E appeared to correspond to a livestock contamination origin.

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium ; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron , certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.

Many countries use wastewater for irrigation. The World Health Organization established, as reuse guidelines, a maximum value of 1 helminth ovum/L for irrigation. Various techniques for enumerating helminth ova in water have been published. To determine the most adequate method for Mexico, four techniques were compared: the U.S. Environmental Protection Agency (U.S. EPA), membrane-filter, Leeds I, and Faust. Two types of water were used: drinking water and municipal wastewater effluent. Sensitivity, discrimination coefficients, precision, recovery efficiency, and cost were determined. In addition, several unseeded wastewater samples were analyzed. For drinking water, U.S. EPA and the membrane-filter techniques demonstrated comparable results; however, when wastewater was used, the membrane technique showed some deficiencies. Because the U.S. EPA technique may be used for samples with both high and low solids content, allows for the recovery of helminth ova with different specific gravities, and has the lowest total cost, it was selected as the best technique.

International audience; The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.

Purification methods for Cryptosporidium oocysts are usually selected on the basis of recovery yield, but the amount of particulate debris in environmental matrices could limit efficiency of oocyst detection by microscopic examination or PCR detection. Previous studies have shown that the standard immunomagnetic separation (IMS) procedure would not be the most suitable method for oocyst purification from turbid matrices. We compared the capacity of Percoll-sucrose flotation and six other density-based purification methods to achieve selective separation of Cryptosporidium oocysts from particulate debris. Rate of oocyst recovery and particulate loading in the purified suspensions were chosen as comparison criteria for the different purification methods. In most earlier studies, the chemical treatments employed to obtain a purified oocyst suspension modify the surface properties of oocysts in spiked samples. Assuming this produces unrealistic conditions affecting the evaluation of purification methods, we performed the present study with native oocysts. Flotation and gradient procedures were tested with and without formaldehyde ethyl acetate (FEA) separation. FEA separation was found to be unsuitable. Filtration and Percoll gradient did not allow selective oocyst separation from debris. Among the purification methods suitable for routine microscopic examination, Percoll-sucrose flotation provided the best recovery rates. For automated enumeration systems or PCR detection, potassium bromide and especially Nycodenz gradients appeared to be the most suitable purification methods. Potassium bromide and Nycodenz gradients provided the best balance between oocyst recovery and particulate load.

This study was performed on sludge samples from 20 wastewater treatment plants located in the north west of France with capacities of 1,000-20,000 inhabitant equivalents. The types of treatment studied were activated sludge low charge with and without denitrification. Respectively, 110 samples of fresh sludge and 84 samples of discharged sludge for spreading were analysed. Globally 78.6% of samples contained helminth eggs belonging to the cestodes (6.1%) and nematodes (93.9%). Most of the nematode eggs detected were viable with 135 positive samples. The distribution, according to genera, indicated a high prevalence of Toxocara eggs (77.4%) followed by Capillaria (13.2%), Trichuris (8.1%) and Ascaris (1.3%). For viable nematode eggs, the concentrations detected ranged from1 to 28/4 gDM for fresh sludge and from1 to 9.6/4 gDM for discharged sludge.

Wastewater Treatment Protozoa Found in Wastewater Influence of Wastewater Treatment on Protozoa Concentrations Protozoa Found in Sludge and the Impact of Treatments Keywords: protozoan parasites; protozoa in wastewater treatment plants; wastewater treatment plants

International audience; The aim of this work was to evaluate the use of molecular techniques for the detection of viable Giardia cysts in the environment to assess public health issues. Three target genes were selected: the heat shock protein gene, HSP70, which is expressed in response to stress; the giardin gene, which encodes a structural protein; and, alcohol dehydrogenase E (ADHE), a novel gene encoding an enzyme involved in the metabolism of energy. We tested the efficiency of five protocols for the extraction of either genomic DNA or total RNA from Giardia cysts: two of these protocols were previously cited in the literature and three consisted of commercial DNA extraction kits. The brands of enzyme were determined according to the primers chosen and the amplification conditions were optimised: 2.5 mM MgCl2, 0.5 mM primers and 60°C for annealing temperature. A semi-nested PCR method and an RT semi-nested PCR procedure were developed to detect mRNA from these three genes and to estimate the viability of Giardia cysts.

International audience; Wastewater treatment results in the production of large quantities of sludge. Agricultural use is an interesting alternative provided that the health risks are taken into account. In France, a decree (1998) defines “sanitized” sludge as sludge that has been treated so that pathogens (Salmonella, enteroviruses, viable nematode eggs) are no longer detectable. The aim of this study was to determine the impact of various sludge treatments on 2 types of pathogens (Salmonella and nematode eggs). We also assessed the levels of micro-organisms that indicate faecal contamination (E. coli, Enterococci, sulfite-reducing anaerobes spores). Various treatment processes were analyzed: 4 biological, 3 chemical, 1 heat , 2 storage and 4 sampling campaigns were carried out. Concerning the parasitological contamination of sludge at the entrance of all sites, concentrations of total nematode eggs and viable nematode eggs were highly heterogeneous (respectively, from 2 to 53 eggs/10 g DM and from 2 to 45 eggs/10 g DM) with viable eggs present in all samples. The total concentration of nematode eggs in the sludge was not greatly affected by treatment or storage, and was in the range

The purpose of this study was to improve understanding of the potential for transfer of the protozoan pathogen Cryptosporidium parvum through aquifers to drinking water wells. Therefore, the factors characterising this transport were experimentally determined. We have developed a continuously recirculating column assay. The latter allows small amounts of C. parvum oocysts to be manipulated providing as much protection as possible from the risks of contamination. As the analysis of oocyst samples is time consuming, a numerical model, simulating the transport phenomena of oocysts under the experimental conditions of assays, was developed to establish the whole experimental curve of results using a small number of experimental points. The comparisons drawn between analytic solutions, experimental results with tracer (NaCl solution) and numerical simulation were in good agreement. A continuously recirculating column assay was performed using oocysts in suspension (flow rate = 1.43 mL/min). Treated sand was used as previous experiments had shown that no adsorption occurs. We observed almost total filtration (99.85%). To check this result, an assay with an open column was carried out under the same conditions. We observed a filtration value of 97%. Consequently, we may say that the continuously recirculating column assay provides satisfactory results.

The cell surface properties of Ascaris suum eggs were investigated to determine the hydrophobic properties of eggs of different origins. Hydrophobic potential measurements were based on adhesion to hydrocarbons. Freshly laid eggs with or without the external coat and eggs that had been stored for 3 months at -20 degrees C were tested. The hydrophobic potential differed according to the hydrocarbon used (hexadecane or octane) and the eggs tested (stored or fresh). The hydrophobic potential with octane was 95% for fresh eggs and 72% for eggs stored at -20 degrees C. Tests with Ascaris eggs with and without an external coat showed similar hydrophobic characteristics; the presence or absence of this external structure did not appear to influence hydrophobic interactions. Acidic interactions also appeared to play no part in the adhesion of eggs to the surface of octane emulsions.

The efficiency of sludge disinfection by irradiation was investigated using an electron beam accelerator, with the Ascaris ovum as a model. Ova suspensions prepared by worm dissection, immediately after preparation and after storage at 4 degrees C for 2 months were tested. Suspensions of ova extracted from slaughterhouse sludge were also tested. The ova were irradiated in sludge to determine, by probit analysis, the dose that inactivated 90% of viable ova. The D10 values obtained for irradiation of residual sludge contaminated with ova depended on the source of the ova, the D10 values were 788 +/- 172 Gy for suspensions of ova extracted from slaughterhouse sludge and 1125 +/- 145 Gy for suspensions freshly prepared by dissection. Ova suspensions freshly prepared by dissection were more proof against irradiation. Similarly, the D10 value was affected by storage: 1125 +/- 145 Gy for freshly produced ova suspensions and 661 +/- 45 Gy for suspensions of ova stored for 2 months at 4 degrees C in deionized water. The medium in which the ova were irradiated (deionized water or sludge) also affected D10 values, the indirect effects were smaller in samples of contaminated sludge, which were rich in organic matter, with the action of the radiation being mostly direct.

Rainwater runoff in urban areas removes helminth eggs into the sewer system where they are carried to the treatment plant by the rainwater or remain in the sewer system until the next heavy rainfall. The concentration of parasite eggs varies according to the water flow rates in the urban system. Heavy rain cleans both the urban surfaces and the drains. In particular, it removes particles less than 100 μm diameter which are laid down in the pipes. These particles are present in high concentration in storm water whenever heavy rain is preceded by days with low rainfall. Variations in parasite concentration are thus dependent upon the precipitation of the previous days making it difficult to predict the concentrations of parasites in raw wastewater at input to the treatment plant. Many factors may affect parasite concentration including the presence of sediment in the sewer system, the intensity of rainfall and the amount of rain that has fallen on previous days. Parasites account for only a small proportion of the mass of suspended solids in rainwater. However, they represent a major health hazard in case of overflow of storm water associated with wastewater into the natural environment.

The major advantage of microfiltration in water treatment is the ability to clarify and decontaminate water in one step. A pilot tangential microfiltration system (a tubular ceramic membrane porosity 0.2μm, surface area 0.135m2) was tested for Cryptosporidium removal. Samples (2201) of artificially contaminated river water were filtered and a removal rate >4.3 log was obtained in nine tests. The viability of rejected oocysts was simultaneously conducted as it is important to be aware of the infectious potential of microfiltration concentrates produced and discharged into the environment. Oocyst viability was unaltered in the rejected concentrates after a normal filtration cycle. However, the process of membrane unclogging by sequential washing with NaOH and HNO3 proved to significantly decrease the number of surviving oocysts in rejected water. Indeed, the washing solutions collecting with the filtration concentrates before disposal resulted in a significant decrease of the viability of oocysts measured by inclusion/exclusion of fluorogenic vital dyes (DAPI and PI).

Helminth eggs (Ascaris, Taenia...) present in urban sludge constitute a sanitary risk when used for the enrichment of agricultural soil. These eggs are very resistant in the environment and their survival could reach 6 months to one year in soils. To control the risks for the public health, we have to determine parameters leading to the eggs destruction in soils. Then the aim of the work is to study several conditions (humidity, temperature, texture,...) that could influence the survival of intestinal nematode eggs in various types of soil. Survival experiments were carried out in clayey soil, silty-loam soil and sandy soils with humidity levels corresponding to: field capacities, wilting point and variation between this 2 limit point (storage temperatures 4 degrees C, 19 degrees C and 30 degrees C). The result of this study has shown that the temperature conditioned for a great part the survival of the eggs in all types of soils with survival times superior at two year with a temperature of 4 degrees C. Between survivals at 20 degrees C and 30 degrees C, no difference has been observed. The humidity is the second parameter that condition the egg survival with a good viability preservation at the wilting point. This study also shows better survival in the conditions of deep soil with the protection of the egg from drying. With these data and with the simulation of continental temperate conditions, a contamination rate of 0.35% could be found on the surface soil after one year and a more important rate in the deep soil with 10.6%. The last step is the validation of this model and calculated values seem to be a good evaluation of the results found on fields. All these data must be take into account for the establishment of the regulation about the urban sludge reused for agricultural purpose.

In the framework of agricultural wastewater reuse, the W.H.O. has defined a parasitological quality for sewage with less than one nematode egg per liter. The purpose of this work is to define an effective method to detect helminth eggs in wastewater. Seven techniques have been applied to waste water analysis, with a comparison of their respective results, varying from 26 to 74 %. Be it in the framework of artificial contamination or on site, the best results were obtained with the diphasic technique perfected at the laboratory including a treatment with antiformine at 8 % + ethylacetate followed by a flotation with zinc sulphate at 55%. The validation in the laboratory of the methods performance on treated wastewater allowed us to show that the yield of the method is significantly independent of the egg concentration as well as giving good homogeneity of results with a concentration of 1 egg/litre.

In third world countries, the agricultural use of treated wastewater represents an interesting solution. A recent report of W.H.O. shows the importance of strict parasitological criteria for such reuse. The aim of this paper is to study the conditions under which Ascaris eggs have been recovered from artificially contaminated soils (sandy, clay or loamy soil, mould garden). The eggs elution has been carried out from soil particles using various solutions (detergents, distilled water, formaldehyde, sodium hydroxide, sodium hypochlorite). The recovery percentage analysis showed the superiority of the sodium hypochlorite solution titrating 10 chlorometric degrees, whatever the soil type. The eggs concentration in the eluates has been carried out through the flotation technique by testing various reagents with densities ranging from 1.16 to 1.44. The zinc sulphate solution at 50%, 55% and 66% prove to be flotation agents that are well adapted to this sampling type. By carrying out the elution with a sodium hypochlorite solution titrating 10 chlorometric degrees and the concentration by flotation with a zinc sulphate solution at 55% the recovery percentages, which are independent of the parasitic load, vary from 66 to 78%.

Recemment, l'attention a ete attiree sur l'importance des criteres parasitologiques dans l'evaluation de la qualite des eaux usees et des boues. Dans ces conditions, des methodes de quantification couplees a des strategies d'echantillonnage bien definies sont necessaires. Dans cette optique, quatre methodes de flottation ou de sedimentation ont ete evaluees, comportant une etude des differents parametres susceptibles d'affecter l'echantillonnage : jour et lieu de prelevement, type de boues. Les effets possibles de ces parametres ont ete analyses a l'aide d'un plan mixte a trois niveaux et repetitions binaires. Enfin, les performances des diverses methodes ont ete testees vis-a-vis de la charge parasitaire, apres contamination artificielle, les autres parametres etant places sous contraintes. Les resultats de l'etude ont permis de selectionner la methode la mieux adaptee en fonction du niveau de contamination et du type de boues.

Two methods of concentration of Salmonella from surface waters were compared. The results obtained show that glass microfiber filters are equivalent to gauze pads for the isolation of Salmonella (32 % yields compared to 23 %). The calculated factor of fecal coliform concentration effectiveness with gauze pad method is never constant (between 1 and 300) and then the method is only qualitative. On the contrary the concentration of Salmonella on the microfiber glass filters pH 3.5 is easily achieved on a punctual sample and, as important, it gives a more reliable, reproductible, quantitative response.

A 1-year survey was undertaken on two biological wastewater treatment plants by two different laboratories, in order to gather information on the enteric viral population concentration and on the removal efficiency of the treatment. Different sampling techniques were compared. Results were comparable and showed that enteric viruses were present all year round, their numbers fluctuating randomly within a day. Composite sampling over a period of at least 15 h gave a good representation of the average virus population.

In countries with limited water resources, the agricultural use of treated waste water represents an interesting alternative. An international report recently‐published shows the importance of strict parasitological criteria for such reuse recommending a limit of less than one viable intestinal nematode egg per liter. This study proposes an experimental method which associates both the detection of very low numbers of Ascaris eggs and their viability determination from samples of wastewater.

A parasitological study was made of different stages of‐ treatment and post‐treatment sludge from a wastewater treatment plant: during treatment, after storage and during composting. Out of 157 samples, 69 % were positive for Nematode eggs and/or Cestode eggs. Anaerobic digestion of sludge had little effect on helminth egg recovery but lime and ferric chloride treatment reduced the number of eggs found in sludge samples Samples of sludge stored for 1 or 2–3 years were all positive but after 4 years of storage, no more helminth eggs were found. After 120 days composting helminth eggs were still recovered. Although compost and stored sludge were positive, no Ascaris eggs tested showed any indication of viability.

Une etude effectuee sur le site de Barcelone a permis de mettre en evidence :1. La tres grande quantite de contaminations microbiologiques (bacteries, virus, oeufs d'helminthes) apportees par les eaux de rivieres, les eaux usees et les boues rejetees en mer.2. La faible concentration en enterovirus dans les sediments marins.3. L'importance des oeufs d'helminthes dans la contamination des sediments et le comportement different des oeufs de cestodes et de nematodes en milieu marin.4. L'impact apparement faible du rejet des boues en mer sur la contamination des sediments.

Different types of sewage sludge produced during wastewater treatment and after storage of up to 5 years were analyzed for both virus and parasite eggs. Among the 145 samples examined, 87.6 % were positive for either Nematode and/or Cestode eggs. Anaerobic digestion of the sludge had little influence on the numbers of helminth eggs while conditioning with lime plus ferric chloride reduced the egg recovery. Helminth eggs were found in sludge samples stored for 1–2 or 3 years, but none were found in samples stored for 4 years or more. None of the Ascaris eggs tested were viable. Out of the 57 sludge samples examined for virus, all the biological sludges contained enterovirus, while only 44 % of the digested sludge samples and 10 % of the semi-dehydrated and conditioned (lime plus ferric chloride) sludge samples were positive for enterovirus. No virus were found in stored sludge. Epidemiological studies are necessary in order to determine any risks presented to public and animal health by the agricultural use of the sewage sludge.

Des prelevements systematiques d'echantillons d'eau destines a l'analyse microbiologique ont permis de definir une zonation du reseau de distribution publique de la ville de Metz. Pour chaque campagne de prelevement (Janvier, Avril et Juin 1984) 120 souches bacteriennes ont ete selectionnees par tirage au prorata de densite de microorganismes isoles par etalement sur milieu gelose nutritif R2A (Reasoner et Geldreich, 1979). Les bacilles gram negatif ont toujours represente groupe predominant. Cependant de Janvier a Juin 1984, leur proportion a diminue d'une maniere sensible (de 91,3 % a 68,1 %) au profit du groupe des bacilles gram +(variation de 7,8 % a 29,2 %). Ce glissement peut etre attribue a une diminution relative des populations de Pseudomonas et une augmentation des populations de Corynebacterium (alors que les bacteries du genre Flavobacterium semblent rester quantitativement stables). La repartition des genres bacteriens au sein du n'est pas homogene et certaines zones apparaissent dominees particulierement les Corynebacterium, etc... Le reseau de distribution publique represente ainsi un enorme reacteur heterogene dont les differentes zones se comportent d'une maniere quasi independante, meme en ce qui concerne la diversite des populations bacteriennes.

This investigation examines the sources of parasite contamination and their effect on the contamination of three types of marine sediments of a specific coastal region: marine sediments, river sediments, and sewage sludge. In the sludge, concentrations varied between 2 × 10 1 to 3.4 × 10 2 eggs kg −1 with 30% of samples containing at least four different types of eggs (nematode and cestode eggs). In the river sediment, helminths eggs were found in 90% of samples (sector Besos) and in 80% of samples (sector Llobregat). Only 20% of the littoral marine sediments from the Besos were positive for helminth eggs but in the sludge effluent zone all the samples were positive. At 7000 m offshore no samples contained helminth eggs. In the Llobregat sector, 35% of samples contained helminth eggs; there was no apparent difference in the degree of parasite contamination between the littoral (200 m offshore) and the intermediate zones (1000 m offshore). At 4000 and 12 000 m offshore no samples contained helminth eggs. The viability of Ascaris eggs was studied. Globally the percentage of viable eggs was always higher in Besos sector than in Llobregat. If all sediment river samples contained viable eggs we have recognized in some sediment marine sample viable Ascaris eggs: in Besos sector 14% of viable Ascaris eggs were still found at 3000 m and 4000 m offshore while only samples collected from littoral zone in Llobregat sector contained 5% of viable Ascaris eggs.

In this study, we have investigated the effect of lagoon treatment on the elimination of helminth eggs. The Marrakech treatment lagoon receiving only part of the city sewage, consists of two connected ponds (total retention time from 8 days to 30 days). Water samples (surface and deep) and sediment samples were collected. In the surface water samples collected at the lagoon entrance there was an average of 11.7 eggs/l, while no helminth eggs could be recovered at the exit from the lagoon. In the two ponds very few eggs were found, both in surface and deep water samples (͌ 3/1). Helminth eggs were mostly found only in the sediment samples: 275 eggs/100 g sediment from the first basin and 158 eggs/100 g sediment from the second, on average. In conclusion, this study demonstrates that lagoon treatment is efficacious in removing helminth eggs. Nonetheless sediments from such treatment present a problem as they remain heavily contaminated with helminth eggs.

After having analyzed several surveys on natural inactivation of viruses in water, the authors have attempted to determine the survival life of poliomyelitis virus type 1 in hydrous medium, of known composition, well-buffered and devoid of any bacterial contamination on the one hand, and on the other hand, in a similar medium with addition of organic humate-type substances. The experiment was carried out at 4 degrees C and at laboratory room temperature (18 degrees C-24 degrees C). Results tend to confirm that the presence of humate-type substances seems to have no significant influence on the development of poliomyelitis virus type 1. Moreover, under the experimental conditions used, the survival life of the virus is much longer than the one previously described. It represents an average of 296 days for a storage temperature of 18 degrees C-24 degrees C. It is longer in water kept at 4 degrees C; in fact, according to statistical calculations and to titrations carried out over a period of 550 days, the total disappearance of the virus was detected only after a period of 10 to 15 years.

Agricultural use of waste water has been regulated mostly by its bacteriological quality. Recently the importance of parasitological criteria for waste water standards was recognized by establishing that waste water containing less than 1 intestinal Nematode egg per liter is released for unrestricted use of effluents. At present, most of the techniques borrowed from parasitological coprology are only qualitative. In this report different protocols for helminth egg quantitation were compared. Three flotation techniques (Janeckso-Urbanyi, Faust, Arther) and two sedimentation techniques (Bailenger, Ritchie) and their performances were evaluated. Although all the techniques were based on the same number of on the whole positive samples, significant differences were observed in both the number of species identified and in the type of egg preferentially concentrated. Among the flotation methods tested, the Janeckso-Urbanyi modality offers undeniable advantages in efficiency and in the variety of species concentrated, but it is relatively costly. Among the sedimentation techniques, the one described by Bailenger is relatively inexpensive and concentrates all the types of eggs normally found in waste water samples, except for the relatively rare Enterobius with globally satisfactory yields.